Project Summary The packaging of the genome into chromatin that is essential for normal growth, development, and differentiation has been a focus of the lab for more than 20 years. Nucleosomes are the basic repeating unit of the chromatin structure that tightly regulates all of the processes that use DNA as a substrate, including transcription, DNA replication, DNA repair, and recombination. The intricate steps of nucleosome assembly are guided by histone chaperones that are critical to prevent the aberrant nonspecific interactions of histone proteins. The key proteins responsible for replication-dependent assembly of nascent histone H3 and H4 are the H3/H4 histone chaperones, Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor (CAF-1). The proliferating-cell nuclear antigen (PCNA) targets chromatin assembly to sites of newly replicated DNA. Our biophysical and structural studies have revealed unexpected, novel and fundamental insights into the early stages of replication-dependent chromatin assembly, namely the hand-off mechanism involving transfer of dimers of H3/H4 from Asf1 to CAF-1, and the architectural features of CAF-1 interactions with H3/H4. While the general functions of these histone chaperones in nucleosome assembly are known, the fundamental molecular and structural mechanisms for these functions remain obscure. As such, this proposal explores questions about important and understudied aspects of H3/H4 histone chaperone activity from the recruitment and mechanism of action at sites of DNA replication which impact the fidelity of epigenetic inheritance as well as a newly-discovered role in the assembly of single-stranded nucleosomes. To answer these questions, biophysical and structural approaches, which have been specifically developed to study histone chaperones will be used together with complementary biochemical and molecular biology approaches in cells. The outcomes of these studies will be a better understanding of the structural mechanisms involved in the process of deposition of H3/H4 onto DNA during nucleosome assembly, and these new insights may inform how chromatin landscapes are established and how the histone chaperones might be manipulated for the purpose of epigenetic regulation in medical research applications and human disease.